Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.26.4 (RNase H)
2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A recombinant p66 form of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) can be obtained [(1991) Biotechnol. Appl. Biochem. 14, 69-81] from crude Escherichia coli extracts by immobilized metal affinity chromatography (IMAC). We have analyzed the p66 HIV-1 RT, isolated in the presence of 0.3 M imidazole, by gel permeation HPLC on Superose 12. The results show that it contains two major distinct p66 forms (24.1 min and 28.3 min peaks) which are distinguishable from the purified homodimeric (p66/p66) HIV-1 RT (22.2 min peak). Protein peak 1 (24.1 min) is converted to a 22.3 min peak upon storage for 20 h at 4 degrees C. Under identical conditions, the isolated peak 2 (28.3 min) appeared as a conformationally heterogeneous mixture elaborated by peaks at 22.3 min and 25.9 min. The protein species thus obtained were active in the RNA-dependent DNA polymerase and RNase H activity assays and produced heterodimeric HIV-1 RT upon incubation with the HIV-1 protease. When the IMAC-purified, imidazole-free homodimeric (p66/p66) form of the enzyme was incubated with 0.3 M imidazole for 16 h at 4 degrees C, protein peaks at 28.3 min (peak A) and 30.5 min (peak B) were isolated by gel permeation HPLC. While both of these p66-containing species were stable and displayed identical RNA-dependent DNA polymerase activities, the protein in peak B was only 50% active in RNase H function compared with the protein from peak A. These imidazole-mediated dissociation studies support the hypothesis of partial unfolding of one of the RNase H domains of the p66/p66 homodimer, suggesting that the p66 subunits are asymmetric in the native enzyme.
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PMID:Human immunodeficiency virus type 1 (HIV-1) recombinant reverse transcriptase. Asymmetry in p66 subunits of the p66/p66 homodimer. 751 87

The reverse transcriptase from human immunodeficiency virus type 1 is a heterodimer consisting of one 66-kDa and one 51-kDa subunit. The p66 subunit contains both a polymerase and an RNase H domain; proteolytic cleavage of p66 removes the RNase H domain to yield the p51 subunit. Although the polymerase domain of p66 folds into an open, extended structure containing a large active-site cleft, that of p51 is closed and compact. The connection subdomain, which lies between the polymerase and RNase H active sites in p66, plays a central role in the formation of the reverse transcriptase heterodimer. Extensive and very different intra- and intersubunit contacts are made by the connection subdomains of each of the subunits. Together, contacts between the two connection domains constitute approximately one-third of the total contacts between subunits of the heterodimer. Conversion of an open p66 polymerase domain structure to a closed p51-like structure results in a reduction in solvent-accessible surface area by 1600 A2 and the burying of an extensive hydrophobic surface. Thus, the monomeric forms of both p66 and p51 are proposed to have the same closed structure as seen in the p51 subunit of the heterodimer. The free energy required to convert p66 from a closed p51-like structure to the observed open p66 polymerase domain structure is generated by the burying of a large, predominantly hydrophobic surface area upon formation of the heterodimer. It is likely that the only kind of dimer that can form is an asymmetric one like that seen in the heterodimer structure, since one dimer interaction surface exists only in p51 and the other only in p66. We suggest that both p51 and p66 form asymmetric homodimers that are assembled from one subunit that has assumed the open conformation and one that has the closed structure.
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PMID:Structural basis of asymmetry in the human immunodeficiency virus type 1 reverse transcriptase heterodimer. 751 28

"BcgI cassette" mutagenesis was used to prepare variants of p66 human immunodeficiency virus (HIV)-1 reverse transcriptase with amino acid substitutions between residues Glu224 and Trp229. Mutant polypeptides were reconstituted in vitro with wild type p51 to generate the "selectively mutated" heterodimer series p66(224A)/p51-p66(229A)/p51. Purified enzymes were characterized with respect to dimerization, DNA polymerase, RNase H, and tRNA(Lys-3) binding. The combined analyses indicate that while alteration of p66 residues Glu224-Leu228 has minimal consequences, the DNA polymerase activities of mutant p66(229A)/p51 are impaired. DNase I footprinting illustrates that this mutant does not form a stable replication complex with a model template-primer. In vivo studies indicate that the equivalent mutation eliminates viral infectivity, suggesting a contribution of Trp229 toward architecture of the p66 primer grip.
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PMID:Mutating the "primer grip" of p66 HIV-1 reverse transcriptase implicates tryptophan-229 in template-primer utilization. 752 8

The p66/p51 human immunodeficiency virus type 1 reverse transcriptase is a heterodimer with identical N-terminal amino acid sequences. The enzyme contains two polymerization domains and one RNase H domain, which is located at the C-terminus of the p66 subunit. Both polymerization domains fold into four individual subdomains that are not arranged in a similar fashion, forming an unusually asymmetric dimer. The complexity of the RT p66/p51 heterodimer structure is simplified using solvent-accessibility surface areas to describe the buried surface area of contact among the different subdomains. In addition, the RT/DNA contacts in the recently published RT/DNA/Fab structure [Jacobo-Molina et al., Proc. Natl Acad. Sci. USA, 90, 6320-6324 (1993)] are described using the same approach. Finally, the RT/DNA complex is compared with other dimeric DNA-binding proteins. It was found that the size of the protein and the extent of the dimer interface were not directly related to the extent of contact between the protein and the DNA. Furthermore, RT, the only protein that is not a sequence-specific DNA binding protein in this analysis, had the largest surface of interaction with the nucleic acid.
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PMID:Buried surface analysis of HIV-1 reverse transcriptase p66/p51 heterodimer and its interaction with dsDNA template/primer. 753 20

The crystal structure of the reverse transcriptase (RT) from the type 1 human immunodeficiency virus has been determined at 3.2-A resolution. Comparison with complexes between RT and the polymerase inhibitor Nevirapine [Kohlstaedt, L.A., Wang, J., Friedman, J.M., Rice, P.A. & Steitz, T.A. (1992) Science 256, 1783-1790] and between RT and an oligonucleotide [Jacobo-Molina, A., Ding, J., Nanni, R., Clark, A. D., Lu, X., Tantillo, C., Williams, R. L., Kamer, G., Ferris, A. L., Clark, P., Hizi, A., Hughes, S. H. & Arnold, E. (1993) Proc. Natl. Acad. Sci. USA 90, 6320-6324] reveals changes associated with ligand binding. The enzyme is a heterodimer (p66/p51), with domains labeled "fingers," "thumb," "palm," and "connection" in both subunits, and a ribonuclease H domain in the larger subunit only. The most striking difference between RT and both complex structures is the change in orientation of the p66 thumb (approximately 33 degrees rotation). Smaller shifts relative to the core of the molecule were also found in other domains, including the p66 fingers and palm, which contain the polymerase active site. Within the polymerase catalytic region itself, there are no rearrangements between RT and the RT/DNA complex. In RT/Nevirapine, the drug binds in the p66 palm near the polymerase active site, a region that is well-packed hydrophobic core in the unliganded enzyme. Room for the drug is provided by movement of a small beta-sheet within the palm domain of the Nevirapine complex. The rearrangement within the palm and thumb, as well as domain shifts relative to the enzyme core, may prevent correct placement of the oligonucleotide substrate when the drug is bound.
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PMID:The structure of unliganded reverse transcriptase from the human immunodeficiency virus type 1. 753 6

The properties of recombinant p66/p51 human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) containing C-terminal truncations in its p66 polypeptide were evaluated. Deletion end points partly or completely removed alpha-helix E' of the RNase H domain (p66 delta 8/p51 and p66 delta 16/p51, respectively), while mutant p66 delta 23/p51 lacked alpha E' and the beta 5'-alpha E' connecting loop. Although dimerization and DNA polymerase properties of all mutants were not significantly different from those of the parental enzyme, p66 delta 16/p51 and p66 delta 23/p51 RT lacked ribonuclease H (RNase H) activity. In contrast, RT mutant p66 delta 8/p51 retained endonuclease activity but lacked the directional processing feature of the parental enzyme. Despite retaining full endoribonuclease function, p66 delta 8/p51 RT barely supported transfer of nascent (-)-strand DNA between RNA templates representing the 5' and 3' ends of retroviral genome, shedding light on the requirement for the endonuclease and directional processing functions of the RNase H domain during replication.
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PMID:Truncating alpha-helix E' of p66 human immunodeficiency virus reverse transcriptase modulates RNase H function and impairs DNA strand transfer. 753 65

Replication complexes containing wild-type and RNase H-deficient p66/p51 human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) were analyzed by DNase I and S1 footprinting. While crystallography and chemical footprinting data demonstrate that 15-18 bases of primer and template occupy the DNA polymerase and RNase H active centers, enzymatic footprinting suggests that a larger portion of substrate is encompassed by the replicating enzyme. Independent of the position of DNA synthesis arrest, template nucleotides +7 to -23 and primer nucleotides -1 to -25 are nuclease resistant. On both DNA strands, position -20 remains accessible to DNase I cleavage, suggestive of an alteration in nucleic acid structure between exiting the RNase H catalytic center and leaving the C-terminal p66 domain. A model of HIV-1 RT containing an extended single-stranded template and duplex region was constructed on the basis of the structure of an RT/DNA complex. Mapping of footprint data onto this model shows consistency between biochemical and structural data, implicating a contribution from domains proximal to the catalytic centers.
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PMID:An expanded model of replicating human immunodeficiency virus reverse transcriptase. 753 89

We have mapped specific RNA-protein contacts between human immunodeficiency virus (HIV) type I reverse transcriptase (RT) and its natural primer, human tRNA(3Lys), using a site-specific crosslinking strategy. Four different tRNA(3Lys) constructs with a single 32P-labeled 4-thiouridine (4-thioU) residue at positions -1, 16, 36 or 41 were synthesized. After incubation with RT followed by irradiation, crosslinks were localized to either the p66 or p51 subunit of RT by digestion with nuclease and SDS gel fractionation. 4-thioU at position -1 or 16 transferred label to the p66 subunit almost exclusively (> 90%), whereas position 36 labeled both p66 and p51 (3:1). Position 41 yielded no detectable crosslinks. The region of p66 contacted by position -1 of tRNA(3Lys) was localized to the 203 C-terminal amino acids of RT by CNBr cleavage, whereas a 127 amino acid-CNBr peptide (residues 230-357) from both p66 and p51 was labeled by position 36. Functionality of the 4-thioU-modified tRNA(3Lys)(-1) crosslinked to RT in the presence of an RNA but not a DNA template was demonstrated by the ability of the tRNA to be extended. These results localize the 5' half of the tRNA on the interface between the two RT subunits, closer to the RNase H domain than to the polymerase active site, in accord with previous suggestions. They argue further that a specific binding site for the 5' end of the primer tRNA(3Lys) may exist within the C-terminal portion of the p66 subunit, which could be important for the initiation of reverse transcription.
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PMID:Site-specific crosslinking of 4-thiouridine-modified human tRNA(3Lys) to reverse transcriptase from human immunodeficiency virus type I. 754 Jan 37

The stimulatory effect of Mg2+ and Mn2+ on the ribonuclease H (RNase H) functions of HIV-1 reverse transcriptase (RT) has been evaluated using a model 90-nt RNA template/36-nt DNA primer. Wild type enzyme exhibits similar endonuclease and directional processing activities in response to both cations, while RNase H activity (hydrolysis of double-stranded RNA) is only evident in the presence of Mn2+. Enzyme altered at the p66 residue Glu478 (Glu478-->Gln478), which participates in metal ion binding, is completely inactive in Mg2+. However, Mn2+ restores specifically its endoribonuclease activity. In the presence of Mn2+, mutant RT also catalyzes specific removal of the tRNA replication primer, eliminating the possibility of contaminating Escherichia coli RNase H in our recombinant enzyme. However, the efficiency with which mutant RT catalyzes transfer of nascent DNA between RNA templates (an event mandating RNase H activity) is severely reduced. These findings raise the possibility that directional processing activity is required to accelerate transfer of nascent DNA between templates during retroviral replication.
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PMID:Divalent cation modulation of the ribonuclease functions of human immunodeficiency virus reverse transcriptase. 754 83

The reverse transcriptase (RT) of HIV-1 has been mutagenized within the carboxyl-terminal domain which harbors the RNase H. Two amino acids highly conserved among all 14 known RT sequences but not in the bacterial RNase H have been mutagenized resulting in the mutant proteins N494D and Q475E. They were expressed as recombinant proteins, purified, and analyzed for their in vitro properties in comparison to the p66 homodimeric wild-type and a previously described H539N mutant. The N494D mutant closely resembles the wild-type RNase H, exhibits an endonuclease activity and a processive RNase H activity, gives rise to small RNA hydrolysis products, and acts in concert with the RT. The Q475E mutant is more defective and resembles the H539N mutant, exhibits a retarded endonuclease activity and an impaired 3'-->5' processive RNA cleavage activity, gives rise to predominantly larger RNA hydrolysis products, is less processive in the presence of competitor substrate, and is defective in its ability to hydrolyze the polypurine tract and homopolymeric hybrids. Short homopolymeric stretches cause a pausing of the RT of wild-type and mutants which results in a coordinated action of the RNase H. Pausing of the RT correlates with RNase H cleavages about 20 nucleotides behind the point of synthesis. The defects of the mutant enzymes can be interpreted on the basis of the known crystallography data.
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PMID:Enzymatic analysis of two HIV-1 reverse transcriptase mutants with mutations in carboxyl-terminal amino acid residues conserved among retroviral ribonucleases H. 767


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